Fmr1-KO mice failure to detect object novelty associates with a post-test decrease of structural and synaptic plasticity upstream of the hippocampus

Mice with deletion of the FMR1 gene show episodic memory impairments and exhibit dendritic spines and synaptic plasticity defects prevalently identified in non-training conditions. Based on evidence that synaptic changes associated with normal or abnormal memory emerge when mice are cognitively challenged, here we examine whether, and how, fragile entorhinal and hippocampal synapses are remodeled when mice succeed or fail to learn. We trained Fmr1 knockout (KO) and wild-type C57BL/6J (WT) mice in the novel object recognition (NOR) paradigm with 1 h or 24 h training-to-test intervals and then assessed whether varying the time between the presentation of similar and different objects modulates NOR performance and plasticity along the entorhinal cortex-hippocampus axis. At the 1 h-interval, KO mice failed to discriminate the novel object, showed a collapse of spines in the lateral entorhinal cortex (LEC), and of long-term potentiation (LTP) in the lateral perforant path (LPP), but a normal increase in hippocampal spines. At the 24 h, they exhibited intact NOR performance, typical LEC and hippocampal spines, and exaggerated LPP-LTP. Our findings reveal that the inability of mice to detect object novelty primarily stands in their impediment to elaborate, and convey to the hippocampus, sensory/perceptive object representations.

and medial perforant path 32 and the CA1 hippocampal subfield 33 when mice were examined at a steady state. However, another study reported that hippocampal synaptic plasticity and episodic memory were normal when assessed separately whereas CA1 LTP measured in cognitively challenged KO mice resulted stronger compared to wild-type (WT) mice 16 . Whether the CA1 LTP alteration observed in normally performing KO mice associates with dendritic spine remodeling, and if these alterations aggravate in mice exposed to tasks in which they fail to learn, is currently unknown. To bridge this information gap, we exposed KO and WT C57BL/6J mice to Novel Object Recognition (NOR) tests that were run either 1 h or 24 h after the presentation of two identical objects. Following each test, dendritic spines were measured in the lateral entorhinal cortex (LEC) and its CA1 and dentate gyrus (DG) hippocampal targets. Given the key role of LEC in novel object/context recognition 34 and evidence that synaptic plasticity in the lateral perforant path (LPP) is altered in naive KO mice 32 , we also assessed the functional status of LEC projections to the hippocampus by comparing LPP basal synaptic activity and LTP in brain slices from NOR-exposed mice of both genotypes.

Materials and methods
Animals. We used 2/3-month-old male Fmr1 KO mice on a C57BL/6J background (CAT# JAX: 003025) and wild-type C57BL/6J (CAT# JAX:000664) obtained from Charles River (Calco, Italy). Fmr1 KO mice were bred by mating homozygous Fmr1 −/− females and hemizygous Fmr1 −/y males. Age-matched C57BL/6J mice were used as a control. All the methods and related experiments were performed in accordance with relevant guidelines and regulations (European Community Guidelines for Animal Care, Italian DL 26/2014, application of the European Communities Council Directive, 2010/63/EU, FELASA and ARRIVE guidelines), and approved by the Italian Ministry of Health and by the local Institutional Animal Care and Use Committee (IACUC). A total of 37 WT mice and 45 KO mice were used in this study.
Novel object recognition (NOR). The NOR test was carried out in 3-month-old KO and WT mice. Mice in their home cage were transferred to the experimental room and left to acclimate for 1 h to the new environment. NOR testing consisted of three sessions. In the first session (open field exploration), each mouse was placed in an empty squared open field (40 cm on the side) surrounded by 60 cm-high walls and left free to explore it for 10 min. The mouse was returned to its home cage for a 10-min pause during which two identical objects, i.e., glass cylinders of 3 cm in diameter and 10 cm in height, were put in opposite corners of the open field. In session 2 (training), the mouse was placed in the center of the open field and allowed to explore two similar objects (O1 and O2) for 10 min. The mouse was returned to its home cage for a 1 h-pause during which one previously explored object (familiar object: FO) was substituted with a novel object (NO), a multicolored Rubik's cube of 5 cm on the side. In session 3 (testing), the mouse was placed again in the center of the open field and allowed to explore the FO and the NO for 10 min. The NOR test was delivered either 1 h (WT mice, n = 10; KO mice, n = 14) or 24 h (WT mice, n = 13; KO mice, n = 15) after training. Mice were returned to their home cage located in the experimental room immediately after the test. Object exploration was defined as mice sniffing or touching the object with its nose and/or forepaws. All objects were cleaned with a 10% ethanol solution before their introduction in the open field. The time spent exploring each object was recorded during sessions 2 (O1 and O2) and 3 (FO and NO), and the discrimination index, the percentage of time spent exploring each object (FO or NO) divided by the total time spent exploring both objects multiplied 100, was calculated 35 . A discrimination index above 50% indicates a preference for the NO, below 50% a preference for the FO, and 50% no preference.

Identical objects recognition (IOR).
To control that neural changes detected in KO mice following NOR actually depend on a perceived discrepancy between the training (similar objects, O1 and O2) and the test (FO vs NO), additional groups of WT (n = 4) and KO (n = 4) mice were subjected to the same three initial phases of the NOR procedure (acclimatization to the experimental room: 1 h; exploration of the empty open field: 10 min; exposure to the two similar objects-training: 10 min) but were instead re-exposed during the test to the same objects explored during training (IOR test duration: 10 min). As in the NOR procedure, mice were returned to their home cage located in the experimental room immediately after the test.
Dendritic spines analyses. One hour and a half after being returned to their home cage, mice were deeply anesthetized with a cocktail of Zoletil (800 mg/kg) and Rompum (200 mg/kg) and perfused transcardially with 0.9% saline solution. Golgi-Cox staining was performed as previously described 36 . Spine density was measured in apical and basal dendrites of layer II LEC, dorsal CA1 pyramidal neurons, and dendrites of granule cells in the molecular layer of the DG. Neurons were identified with a light microscope (Leica DMLB) under low magnification (20×/NA 0.5) and analyzed under higher magnification (100×/NA 0.5). On each neuron, spines were counted in randomly selected five 30-100 μm dendritic segments of secondary and tertiary branch order of basal dendrites using Neurolucida software. Statistical comparisons were made on single neuron values obtained by averaging the number of spines counted on segments of the same neuron. The analysis was conducted by an experimenter blind to the experimental condition.
Image analysis. Signals for vGlut1 and PSD95 were detected separately from ten non-overlapping ROIs (10 × 10 µm squares) from each image; ROIs were randomly selected avoiding DAPI-labeled cell bodies. IMA-RIS software was used for automatic detection and counting of PSD95 and vGlut1 puncta and identification of PSD95/vGlut1 co-localization as overlapping signals. For all the experiments, ROIs were analyzed after establishing a detection threshold, which was kept constant within each measurement.
Slice preparation and electrophysiology recordings. Mice were sacrificed by cervical dislocation, and the brains were isolated and immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH 2 PO 4 , 1.2 MgCl 2 , 2 CaCl 2 , 25 NaHCO 3 , 11 glucose (pH 7.3) saturated with 95% O 2 and 5% CO 2 . Parasagittal slices (400 μm) containing LEC and hippocampus were maintained at room temperature (22-24 °C) in ACSF for at least 1 h, then, each slice was transferred to a submerged recording chamber and continuously superfused at 32-33 °C with ACSF at a rate of 2.6 ml/min. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the dentate gyrus with a glass microelectrode filled with 2 M NaCl solution (pipette resistance 2-5 MΩ), upon stimulation of lateral perforant path (LPP) with an insulated bipolar twisted NiCr electrode (50 μm OD). Stimulation and recording electrodes were placed in the outer part of the molecular layer of the dentate gyrus 38 , 3-4 mm apart, with the stimulation electrode at the edge of the entorhinal cortex (EC) and the hippocampal fissure. Responses were identified by application of paired-pulse stimulation (PPS, two consecutive pulses 50-ms apart), and only fEPSPs showing paired-pulse facilitation (PPF, R2/R1 ratio ≥ 1) were used for recording. Paired-pulse ratio (PPR) was used as an indicator inversely proportional to presynaptic neurotransmitter release 39 . After PPS, during normal recording, each pulse was delivered every 20 s (square pulses of 100 μs duration at a frequency of 0.05 Hz), and three consecutive responses were averaged. Signals were acquired with a DAM-80 AC differential amplifier (WPI) and analyzed with the LTP program 40 . The input/output (I/O) curves were plotted as the relationship between the fEPSP slope (mV ms −1 ) and the stimulation intensity (µA). LTP was induced by using a high-frequency stimulation (HFS) protocol consisting of two trains of 100 pulses at 100 Hz, 20 s apart. HFS was applied in disinhibited slices (10 μM bicuculline methiodide, Tocris Biosciences, Bristol, UK) to facilitate LTP expression at perforant path synapses 38 .

Statistical analysis.
As for the NOR test, the time (in sec) spent exploring each object category in sessions 2 and 3, were compared in each genotype using two-tailed Student's t-tests for paired samples. In the IOR test, an ANOVA for repeated measures with genotype as main factor and sessions as repeated factor was used to estimate habituation of object exploration across training and test sessions. Object preference indexes were compared between genotypes using two-tailed Student's t-tests for unpaired samples. Spine density scores and expression levels of synaptic proteins were compared using two-way ANOVAs with genotype (WT vs KO) and testing condition (NOR-tested vs naive) as main factors. Post hoc pair comparisons were carried out where necessary using the Bonferroni test. Electrophysiological data were compared using a two-tailed Mann-Whitney test for independent groups, and the Wilcoxon test for pre-vs post-bicuculline treatment comparisons. Statistical analyses and curve fittings were obtained by using Stat 3 software and GraphPad Prism software (version 6.05).

NOR 1 h post-training. KO mice show NOR impairment. KO mice exhibit learning deficits in aversively-
and positively-motivated episodic memory tasks which are subtle 21 or emerge when cognitive demand is high 23 . Compared to these protocols, non-associative spatial 22 or object recognition 22,23 tasks appear more appropriate to detect cognitive dysfunctions as variations in the duration of the sampling phase or the sampling-to-test interval allow to appraise the speed of sensory information processing and the recognition memory span 41 . When testing was delivered 1 h after training, we found that NOR-exposed KO (blue bar) and WT mice (grey bar) similarly explored the identical objects in session 2 ( www.nature.com/scientificreports/ LEC spines collapse in KO mice following NOR testing. Statistical comparisons of LEC spines revealed a significant effect of genotype (F (1,28) = 24,7; p < 0.001), and of the genotype × testing condition interaction (F (1,28) = 13,7; p < 0.001). As shown in Fig. 2A, more LEC spines were counted in KO mice than in WT mice in the naive condition (p < 0.001). In the WT mice, no variation in LEC spines was found between NOR-exposed and naive mice (WT tested vs naive, p = 0.23) whereas a collapse of spines was found in NOR-exposed KO mice which exhibited spine scores below those of their naive counterpart (KO tested vs naive, p < 0.001).
LEC and CA1 synaptic proteins are decreased in KO mice following NOR testing. ANOVAs comparing numbers of immuno-reactive puncta revealed significant genotype × NOR-exposure interactions for vGluT1, PSD95, and vGluT1/PSD95 co-localization (p < 0.01 for all comparisons) in LEC (Fig. 3A) and CA1 (Fig. 3B). In comparison with naive WT mice, naive KO mice exhibited significantly more single-protein (EC and CA1 vGluT1: p < 0.01; EC PSD95: p < 0.01, CA1 PSD95: p < 0.001) and co-localized (EC and CA1: p < 0.001) puncta. NOR-exposed KO mice exhibited a massive decrease in single-protein and co-localized puncta in LEC where we detected a collapse of spines, and, unexpectedly, in CA1 where we detected an increase in spines (p < 0.001 for all comparisons except PSD95 in LEC, p = 0.07). No variation in protein expression was found between NOR-exposed and naive WT mice.
LPP basal synaptic activity is normal in KO mice following NOR testing. PPR did not significantly vary (p > 0.1) between NOR-exposed and naive WT mice (Fig. 4A, WT (Fig. 4E).
Bicuculline-induced disinhibition of LPP synaptic activity is absent in KO mice following NOR testing. Consistently with data showing that application of the γ-Aminobutyric acid type A (GABAA) receptor antagonist bicuculline methiodide (10 µM) potentiates fEPSP and facilitates induction of synaptic plasticity at perforant path synapses 39 , we observed a significant increase in LPP synaptic activity in bicuculline methiodide-treated slices from WT NOR-1 h mice (Fig. 4F, 110.4 ± 3.9% of basal slope, p < 0.05 vs pretreatment basal level) that was not present in KO NOR-1 h mice (Fig. 4G, 99.4 ± 1.9% of basal slope, p > 0.1 vs pretreatment basal level) consistently with the reduced GABAergic inhibitory tone in their hippocampus [42][43][44] .
LEC and CA1 synaptic proteins do not vary between genotypes following NOR testing. The same pattern of synaptic proteins expression was observed in LEC (Fig. 3C) and CA1 (Fig. 3D). There was a significant genotype × NOR exposure interaction for vGluT1, PSD95, and VGluT1/PSD95 co-localization in LEC and CA1 (p < 0.05 for all comparisons). In each region, the level of each protein and their co-localization were higher in naive KO mice than in naive WT mice (p < 0.01 for all comparisons). These levels were further increased in WT tested mice compared to naive WT mice (LEC: vGluT1, p < 0.05; PSD95 and vGluT1/PSD95, p < 0.01; CA1: vGluT1, PSD95 and vGluT1/PSD95, p < 0.05 for all comparisons). Differently, NOR-exposed KO mice did not show any increase in synaptic proteins since their levels were in the same range as those of NOR-exposed WT mice (p > 1 for all comparisons).

Discussion
Consistent with the view that memory impairments in FXS [20][21][22][23] or other mouse models of intellectual disabilities 45 need more time to learn, we found that KO mice fail to detect object novelty when the training-to-test interval is fixed at 1 h whereas they succeed when the interval is extended to 24 h. It is therefore apparent that augmenting the time which separates the presentation of similar and different objects does not render the memory of the familiar object weaker but instead facilitates the initial encoding of object similarity as well as subsequent detection of object novelty. Confirming that NOR failure depends on a selective deficit in the perception of a discrepancy between the presentation of similar (training) and different (testing) objects, control experiments in which KO and WT mice were exposed to the same configuration of identical objects during training and testing revealed no genotype difference in the rate of object exploration in any session and robust and similar habituation of objects exploration across sessions. Successful NOR has been shown to depend on close interactions between neurons in perirhinal/lateral entorhinal cortices, which are object detectors 46 , and hippocampal neurons, which compare and identify mismatches between current information vs previously formed environmental representations stored in parahippocampal/neocortical regions 47,48 . By showing that NOR failure is associated with utmost disruption of plasticity upstream the hippocampus and that LEC spines and LPP-LTP plasticity are not merely refractory to experiencedependent changes but collapse when mice are tested 1 h after training, our data reveal that KO mice have a primary impediment in elaborating and encoding representations of similar/different object characteristics. The origin of this impediment likely stands in the defective processing of sensory information extensively reported in FXS individuals and mouse models 49 which has been ascribed to hyperexcitable cells and circuits 50,51 and immature synapses 52 in primary sensory regions. Of note, the similar habituation of object exploration observed in KO and WT mice exposed to the IOR test did not elicit changes in LEC or CA1 spines in any genotype. Specifically, spines did not vary between IOR-tested KO and WT mice although they were still more abundant in KO mice than in WT mice.
Assuming that a distorted sensory input reaches temporal lobe regions, surprisingly, we found that it triggers differential structural remodeling of entorhinal and hippocampal synapses. The paradoxical aspect of differential spine remodeling in LEC and CA1 is that these neurons show the same baseline morphological abnormalitiesabundant immature spines, and exhibit the same form of reactive structural plasticity when exposed to enriched environment stimulation 7,9,53 . Confirming that region-specific differential remodeling is actually triggered by NOR failure, repeated exposure to similar objects during training and testing did not elicit changes in LEC or CA1 spines in any genotype.

Figure 2.
Dendritic spines and representative dendritic segments in entorhinal cortex (LEC) and CA1 pyramidal neurons from naive and NOR-tested KO mice. 1 h interval: Consistently with their immature spine morphology, naive KO mice (grey solid bars) exhibit significantly more spines than naive WT mice (grey empty bars) in both LEC (A) and CA1 (B). Following NOR failure, KO mice (light blue solid bars) show opposite remodeling in LEC and CA1 with a decrease in LEC spines and an increase in CA1 spines relative to their naive siblings (grey solid bars). Differently, in the WT mice, successful NOR does not elicit any change in spine density in any region relative to their naive siblings (NOR-tested WT mice: light blue empty bars; naive WT mice: grey empty bar). Values are expressed as the number of spines per 1 µm segment. LEC: naive WT (n = 10), naive KO (n = 22), NOR-tested WT (n = 8), NOR-tested KO (n = 22); CA1: naive WT (n = 10), naive KO (n = 22), NOR-tested WT (n = 22), NOR-tested KO (n = 24); 24 h interval: Successful NOR in KO mice was associated with a significant increase in LEC spines (C) but no significant change in CA1 spines (D) whereas in WT mice it was associated with significant increase in spines in both regions (NOR-tested KO mice: dark blue solid bars; naive KO mice: grey solid bars; NOR-tested WT mice: dark blue empty bars; naive WT mice: grey empty bars). Remarkably, at this interval where both KO and WT mice show successful NOR the spine scores of NOR-tested KO mice do not significantly differ from those of NOR-tested WT mice. www.nature.com/scientificreports/ Differently, WT and KO mice achieve successfully NOR when a 24 h interval is interposed between training and testing, and exhibit similar spine scores in LEC and CA1. This similarity depends, however, on the fact that exposure to Long-Term NOR brings the density of WT spines to the level constitutively expressed by KO mice. Thus, no elevation of spine density imputable to successful performance is detected in this genotype, consistently with the report that disease-associated spine changes above (autism spectrum disorders) or below (Alzheimer's disease) a physiological threshold alter the functional state neural circuits and the cognitive operations they support 54 . Remarkably, there was a good alignment between variations, or no variation, in spines and synaptic protein levels in this NOR condition. Specifically, NOR-tested WT mice showed increased levels of vGluT1, PSD95, and their co-localization in LEC and CA1, consistently with the augmentation of spines in these regions, whereas NOR-tested KO mice did not show variations in synaptic proteins, consistently with the stability of spines in these regions. Minor deviations from this alignment like the only increase in vGluT1 in the LEC of WT mice, or the decrease in PSD95 in the CA1 of KO mice do not invalidate this central observation. Interestingly, exaggerated induction and magnitude of LPP-LTP, which is observed in the unique condition where KO mice show an increase in LEC spines, remain a peculiarity of NOR exposed KO mice. This phenomenon can be viewed as some form of neural compensation 55 since, because of the immature spines and synaptic protein defects observed in the region, the LPP-LTP overshooting is likely required to drive a sufficient amount of structural remodeling warranting successful NOR.
In the continuous flow of sensory stimuli that every living organism perceives in its environment, the ability to distinguish between new and familiar stimuli is of crucial adaptive importance. Computational 56 and physiological 57,58 models of the mammalian brain posit that the hippocampus is central to novelty detection because its anatomical circuitry is ideally featured to detect mismatches between incoming sensory inputs and previously encoded representations. The prerequisite of these models is that the information which is detected and stored within perirhinal and entorhinal cortices during object exploration is regularly conveyed to the hippocampus by the perforant path. The point is that perirhinal and entorhinal cortices are not simple nodes where sensory information transits before it reaches the hippocampus, but regions where representations of objects are formed and temporarily stored 59 . Thus, the collapse of entorhinal synapses and LPP are expected to mimic the effect produced by disconnecting the entorhinal cortex from the hippocampus 60 to the extent that little specific sensory information regarding objects reaches, and is processed by, the hippocampus. This jeopardizes the function of constitutively fragile, but structurally plastic, nodes downstream of the LEC like CA1 and DG, which fail to support meaningful representations 61 due to the insufficiency of incoming explicit sensory information.
A limitation of the present study might be that a behaviorally induced modulation of neuroplasticity should be confirmed by showing causal inverse relationship between neuroplasticity and behavior. Nevertheless, given the opposite modifications of plasticity detected in LEC vs CA1, attempts to correct the behavioral impairment in KO mice or to generate it in WT mice would require to concurrently perform region-specific opposite (longterm depression and LTP) manipulations whose effects on behavior might be difficult to control and interpret.
As previously emphasized 62 , NOR studies in rodent models of cognitive disabilities have analyzed considerably more memory than perception, affordances, and formation of object representations. Because basic sensory and memory circuits are conserved across mammalian species 49 , we believe that preclinical studies showing how atypical/distorted sensory inputs are processed by entorhinal-hippocampal regions in FXS mice models can shed light on neural mechanisms at the origin of intellectual disabilities in FXS individuals.
In particular, we observed that a short interval inserted between the presentation of similar and dissimilar objects, that does not allow mice to perceive the gap between the two object configurations, is associated with a post-test collapse of LEC synapses. This demonstrates that hemizygous deletion of the Fmr1 gene affects the processing of sensory information more severely than the associative operations, the comparison, and storage of object representations, depend on. Acknowledging the limited translational potential of the present findings, far from the understanding of the neural mechanisms behind learning impairment or improvement in FXS, our results however suggest that rehabilitation strategies combining temporal lobe cortical stimulation with the presentation of learning tasks that systematically facilitate the discrimination and encoding of sensory stimuli might be beneficial for FXS individuals. . Density of immunoreactive spots and representative immunofluorescence images of presynaptic (vGluT1) and postsynaptic (PSD95) proteins in LEC and CA1 from naive and NOR-tested KO mice. (A,B) 1 h interval: Consistently with their higher spine density, naive KO mice (grey solid bars) exhibit more singleprotein and co-localized immunoreactive spots than naive WT mice (grey empty bars) in both regions whereas, in line with their NOR deficit at the 1 h interval, synaptic proteins are decreased in LEC and CA1 of NORtested KO mice (light blue solid bars) compared to naive KO mice (grey solid bars). Differently, and in line with the absence of variations in spine density at the 1 h interval, these synaptic markers do not vary in any region between NOR-tested WT mice (blue empty bars) and naive WT mice (grey empty bars). LEC: naive WT (n = 4), naive KO (n = 4), NOR-tested WT (n = 6), NOR-tested KO (n = 10); CA1: naive WT (n = 4), naive KO (n = 3), NOR-tested WT (n = 6), NOR-tested KO (n = 10). *p < 0.05; **p < 0.01; ***p < 0.001. 24 h interval: In both regions (C,D), naive (grey solid bars) and NOR-tested (dark blue solid bars) KO mice exhibit comparable numbers of vGluT1 and PSD95 immunoreactive spots. Differently, NOR-tested WT mice (dark blue empty bars) showed increased VGluT1 and PSD95 immunoreactivity compared to naive WT mice (grey empty bars). As for spine density measurements, the number of immunoreactive spots for each protein did not differ between NOR-tested WT mice and NOR-tested KO mice. LEC: naive WT (n = 4), naive KO (n = 4), NOR-tested WT (n = 6), NOR-tested KO (n = 9); CA1: naive WT (n = 4), naive KO (n = 3), NOR-tested WT (n = 6), NOR-tested KO (n = 7). *p < 0.05; **p < 0.01; ***p < 0.001.  www.nature.com/scientificreports/

Data availability
The data will be made available from the corresponding authors (Alberto Martire and Martine Ammassari-Teule) upon acceptance for publication on reasonable request.